A number of molecules and specified regions thereof have been implicated in the ability to support the sprouting of neurites from a neuronal cell, a process also referred to as neurite outgrowth. This process is essential in neural development and regeneration. As such, understanding the structure, function and expression of molecule or molecules that mediate, separately or in concert the complex molecular and cellular events in regulating neurite outgrowth in the nervous system is of paramount importance for both diagnostic and therapeutic uses.
Cell adhesion molecules, also referred to as CAM's have been shown to mediate cell to cell interaction in the nervous system in processes critical for embryonic development and pattern formation. See, Edelman, et al., Morphoregulatory Molecules, John Wiley & Sons, New York (1990). Many of the interactions between neural cells with other neural cells, with support cells such a glia or with the extracellular environment involve cell adhesion molecules, as described by Edelman, Science, 219:450-457 (1983).
Several neural cell adhesion molecules have been shown to belong to the immunoglobulin superfamily [Williams et al., Ann. Rev. Immunol., 6:381-405 (1988)] that is characterized by the presence of structural motifs consisting of immunoglobulin-like domains (Ig-like). Many-members of this family are also characterized by the presence of fibronectin type III repeats (Fn type III). Various vertebrate molecules in the Ig family include neural cell adhesion molecule (N-CAM) [Cunningham et al., Science, 236:799-806 (1987)], neuron-glia CAM (Ng-CAM) [Burgoon et al., J. Cell Biol., 112:1017-1029 (1991)], L1 [Kadmon et al., J. Cell Biol., 110:193-208 (1990)], Nr-CAM [Grumet et al., J. Cell Biol., 6:1399-1412 (1991)], neurofascin [Volkmer et al., J. Cell Biol., 118:149-161 (1992) and the like. These molecules have all been shown to be involved in cell adhesion.
Invertebrate members that resemble the vertebrate Ig-like molecules in structure and function include fasciclin II [Harrelson, et al., Science, 242:700-707 (1988), neuroglian [Bieber et al., Cell, 59:447-460 (1989) and the like.
By virtue of the number of domains and amino acid identities among the cell adhesion molecules that are members of the Ig superfamily, subfamilies are grouped based on having the same number of Ig-like domains and Fn type III repeats along with greater amino acid similarity of the members, particularly in their cytoplasmic regions. One such subfamily is characterized by having six Ig-like domains and five Fn type III repeats. Members of this subfamily, referred to as the 6/5 family herein, include chicken Ng-CAM, L1, and chicken Nr-CAM that are all post-translationally cleaved in vivo at comparable sites in the middle of the third Fn type III repeat.
The complete nucleotide and amino acid sequence, along with characterization of the biological activities of some of the 6/5 family members have been described in the literature. Ng-CAM, cloned and sequenced as described by Burgoon et al., J. Cell Biol., 112:1017-1029 (1991), is a membrane glycoprotein of the chicken nervous system that is expressed by neurons and Schwann cells and is involved in neuron-neuron and neuron-glia adhesion. Antibody perturbation studies have indicated that it functions in the fasciculation of neurites and in the migration of neurons along Bergmann glial fibers during cerebellar development [Chuong et al., J. Cell Biol., 104:331-342 (1987) and Hoffman et al., J. Cell Biol., 103:145-158 (1986)]. Ng-CAM promotes cell adhesion both homophilically and heterophilically. The biochemistry and biology of Ng-CAM is reviewed by Grumet, J. Neurosci. Res., 31:1-13 (1992).
The predominant Ng-CAM component detected in chicken brain is a 135 kilodalton (kD) glycoprotein but smaller amounts of a 80 kD glycoprotein and a doublet of 190 and 210 kD (which differ in glycosylation of a single polypeptide) are usually seen as described by Grumet et al., Proc. Natl. Acad. Sci., USA, 81:7989-7993 (1984). All of these components are derived from a single gene and a single mRNA that encodes the larger 190/210 kD species, designated herein as Ng-CAM 200 as described by Burgoon et al., J. Cell Biol., 112:1017-1029 (1991). The smaller components are generated by proteolysis yielding the amino-terminal 135 kD extracellular fragment, designated herein as F135, and the 80 kD transmembrane fragment, designated herein as F80.
F135 and F80 each contain structural motifs that could contribute to the adhesive functions of Ng-CAM. The F135 polypeptide contains all six Ig-like domains, which in N-CAM and other members of the N-CAM family have been demonstrated to mediate adhesion. See, Brady-Kalnay et al., J. Biol. Chem., 269:28472-28477 (1994); Cunningham et al., Proc. Natl. Acad. Sci., USA, 80:3116-3120 (1983) and Rao et al., J. Cell Biol., 118:937-949 (1992). Furthermore, the amino-terminal segment of F80 includes, within the third Fn type III domain, an Arg-Gly-Asp (RGD) sequence that in fibronectin has been demonstrated to mediate adhesion to integrin receptors as described by Ruoslahti, Ann. Rev. Biochem., 57:375-413 (1988).
The complete nucleotide and amino acid sequences of the Ng-CAM homologous molecules, mouse L1 and human L1, have been respectively described by Moos et al., Nature, 334:701-703 (1988) and Hlavin et al., Genomics, 11:416:423 (1991). Variants of human L1 arising through alternate splicing of RNA were described by Reid et al., J. Mol. Neurosci., 3:127-135 (1992). The complete nucleotide and encoded amino acid sequence of the other 6/5 family member, Nr-CAM, have been described by Grumet et al., J. Cell Biol., 113:1399-1412 (1991). While these references discuss the relationship of the particular molecule to the other members of the subfamily, no reference describes polypeptides corresponding to Ng-CAM F80 or the Fn type III repeats spanning the third to fifth repeat (Fn3-5) or the fourth to fifth repeat (Fn4-5) as having neurite outgrowth promoting activity.
However, in other published references, both the Ig domains and the FN type III repeats of N-CAM [Frei et al., J. Cell Biol., 118:177-194 (1992)] and of mouse L1 [Appel et al., J. Neurosci., 13:4764-4775 (1993)] have been postulated to promote neurite outgrowth and spreading of neuronal cell bodies. In the latter publication, in contrast to the present invention, the regions of mouse L1 shown to promote neurite outgrowth were the Ig-like domains 1-6 and the Fn type III repeats 1 and 2, not Fn type III repeats 3-5 as shown. Moreover, Appel et al. showed that the Ig domains 1-2 and 5-6 and Fn type III domains 3-5 of mouse L1 supported neuronal attachment and not neurite outgrowth as demonstrated by the present invention for Fn3-5, Fn4-5 and F80.
In an International Publication having Publication Number WO 95/13291 by New York University, Ng-CAM and its functional derivatives have been claimed as having neurite outgrowth-promoting activity. As published, the functional derivatives of Ng-CAM claimed to have neurite activity were the first three Ig-like domains and not the Fn type III repeats of the present invention.
Thus, while it is well known that the intact Ng-CAM protein, like that of intact L1 protein and other members of the 6/5 family as well as other cell adhesion molecules in other subfamilies, promotes both cell adhesion and neurite outgrowth, what was not appreciated before the present invention was that, in members of the 6/5 family, the region of the fibronectin type III repeats between the third and the fifth repeat, but not including the RGD-containing region.